Gene therapy that results in apoptosis of synovial fibroblasts

Context

A prominent feature of the rheumatoid synovium is the dysregulated hyperplasia of SFs due to a postulated imbalance between growth and apoptosis signals. Stimulation of SFs from RA patients with TNF-a results in enzyme secretion, which may contribute to articular destruction. Induction of apoptosis of this fibroblast population may represent a potential therapeutic approach in RA. Stimulation of cells by TNF-a can generate two signals: one initiates apoptosis, whereas the second leads to activation of NF-?B (which in turn produces inhibitors of apoptosis [IAPs] and promotes the production of pro-inflammatory factors). To induce an apoptotic response in RASFs to TNF-a through adenoviral expression of a truncated stable form of I?Ba and expression of an antisense fragment to XIAP.

Significant findings

AdCMVI?B-DN expressed a truncated form of I?Ba by western blot, that efficiently inhibited TNF-a induced NF-?B nuclear translocation in RASFs. RASFs were resistant to TNFa-mediated apoptosis; however, this resistance was lost upon transduction with AdCMVI?B-DN, associated with caspase 3 activation. In the in vivoSCID mouse model of RASF hyperplasia, extensive apoptosis was only observed in joints that received AdCMVI?B-DN in combination with systemic TNF-a. The degree of bone erosion was similar for all treatment groups. XIAP was shown to be induced in RASFs by TNF-a in a dose-dependent manner through an NF-?B dependent mechanism, since it could be blocked by infection of cells with AdCMVI?B-DN. The contribution of XIAP to the resistance of RASFs to TNF-a apoptosis was determined by infecting RASFs with AdCMVXIAP-AS or the control GFP construct and then exposing the cells to TNF-a (10 ng/ml) for 12 h. RASFs infected with AdCMVXIAP-AS displayed up to 80% apoptosis after TNF-a treatment compared to control cells.

Comments

This interesting study extends the work of Miagov et al, through the use of human rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and in an in vivo SCID mouse model of synovial hyperplasia. Blockade of NF-?B with a stable mutant I?Ba deviates the response of RASFs to tumour necrosis factor (TNF)-a from non-apoptotic to an apoptotic pathway both in vitro and in vivo. These data further support the therapeutic potential of targeting NF-?B. In addition the same effect is achieved in vitro through blockade of the downstream X-linked inhibitor of apoptosis (XIAP). XIAP may represent a more selective apoptosis-inducing target compared with the numerous transcriptional targets of NF-?B; however, its precise functions require further characterisation. Whilst fragments of XIAP are known to inhibit caspase activity, a recent study has shown XIAP can induce NF-?B activation in a regulatory loop. Further in vitro studies on normal synoviocytes and in vivostudies in experimental models of arthritis will clarify the therapeutic potential of targeting XIAP expression/function.

Methods

Primary synovial cell lines were established from tissue obtained from patients undergoing total knee replacement for RA. Adenoviral constructs encoding a mutated I?Ba (AdCMVI?B-DN), an antisense XIAP fragment from -34 to +80 (AdCMVXIAP-AS), green fluorescent protein (GFP) (AdCMVGFP) and LacZ (AdCMVLacZ) were used in this study. The effect of transduction of RASF with AdCMVI?B-DN or AdCMVGFP in vitrowas assessed as follows: nuclear translocation of NF-?B in response to TNF-a was assessed by gel shift analysis; TNF-a-induced caspase 3 activation was determined by western blot; apoptosis in the absence or presence of TNF-a was measured by Hoechst 33258 staining. Injection of RASF intra-articularly in both knees of SCID mice manifests fibroblast hyperplasia after 4 weeks. These fibroblasts were then transduced by inta-articular injection of AdCMVI?B-DN or AdCMVLacZ with or without TNF-a (10 ?g/kg) given systemically 2 days later. Hyperplastic growth was determined histologically by TUNEL staining of tissue obtained 24 h later. The expression of XIAP in RASFs in response to TNF-a was assessed by northern blot and RT-PCR. The effect of blocking XIAP expression, by transduction of RASF with AdCMVXIAP-AS, on TNF-a induced apoptosis was determined by Hoechst staining.